Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells.

Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, Helicobacter pylori infection of the stomach has been correlated with epigenetic changes in the host genome. To identify epigenetic changes during Escherichia coli induced urinary tract infection (UTI), we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC), resulting in intracellular bacteria colonies. Cells inoculated with FimH-negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. UPEC infection significantly induced de novo methyltransferase (DNMT) activity (12.5-fold P=0.002 UPEC vs non-inoculated and 250-fold P=0.001 UPEC vs N-UPEC inoculated cells) and Dnmt1 RNA expression (6-fold P=0.04 UPEC vs non-inoculated cells) compared with controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT-1197) in response to UPEC infection as demonstrated by confocal analysis. Real-time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC-induced downregulation of the tumor suppressor gene CDKN2A (3.3-fold P=0.007 UPEC vs non-inoculated and 3.3-fold P=0.001 UPEC vs N-UPEC) and the DNA repair gene MGMT (9-fold P=0.03 UPEC vs non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8-fold P=0.04 UPEC vs N-UPEC and UPEC vs non-inoculated). Methylation of MGMT was not affected. UPEC-induced methylation of CDKN2A exon 1 may increase bladder cancer and presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence.

Utility of Electronic Medical record-based Fibrosis Scores in Predicting Advanced Cirrhosis in Patients with Hepatitic C Virus Infection.

OBJECTIVE: To determine whether advanced cirrhosis - defined by the detection of nodular liver contours or portal venous collaterals on imaging studies - could be predicted by fibrosis algorithms, calculated using laboratory and demographic features extracted from patients' electronic medical records. To this end, we compared seven EMR-based fibrosis scores with liver imaging studies in a cohort of HCV patients. METHODS: A search of our health system's patient data warehouse identified 867 patients with chronic HCV infection. A total of 565 patients had undergone at least one liver imaging study and had no confounding medical condition affecting the imaging features or fibrosis scores. Demographic and laboratory data were used to calculate APRI, Fib4, Fibrosis Index, Forns, GUCI, Lok Index and Vira-HepC scores for all viremic patients who had undergone liver imaging. Data points selected for the calculation of these scores were based on laboratory results obtained within the shortest possible time from the imaging study. Areas under the receiver operating curves (AUROC), optimum cut-offs, sensitivities, specificities and positive and negative predictive values were calculated for each score. RESULTS: Seven algorithms were performed similarly in predicting cirrhosis. Sensitivities ranged from 0.65 to 1.00, specificities from 0.67 to 0.90, positive predictive values from 0.33 to 0.38, and negative predictive values from 0.93 to 1.00. No individual test was superior, as the confidence intervals of all AUROCs overlapped. CONCLUSIONS: EMR-based scoring systems performed relatively well in ruling out advanced, radiologically-defined cirrhosis. However, their moderate sensitivity and positive predictive values limit their reliability for EMR-based diagnosis.

Pathologic bladder microenvironment attenuates smooth muscle differentiation of skin derived precursor cells: implications for tissue regeneration.

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.